(1) Field of the Invention
The invention concerns the invention and production of mutant RecA homolog proteins by substituting amino acid residues at particular residue positions along the MAW (Makes ATP Work) motif. The MAW motif is defined as amino acid residues 40 to 65 in the Escherichia coli (E. coli) RecA protein (SEQ ID NO: 1) and the homologs of this structure in other proteins. These mutants are classified as Class V, VI, and VII mutants. Class V mutants of this form can be active independently of ATP, which is required to activate wildtype RecA. Class VI mutants of this form can exhibit tighter binding to DNA than wildtype RecA. Class VII mutants exhibit combinations of Class V and VI mutant properties to provide both ATP-independence and tighter DNA binding.
(2) Description of the Related Art
The RecA protein of E. coli is an important player in the in vivo processes of homologous recombination and recombinational DNA repair. Its functions in vivo depend upon several key activities identified in vitro, including the binding of multiple DNA molecules and the hydrolysis of ATP. Such ligand interactions allow the detection of complementary sequences between homologous DNA molecules, generating a Holliday-like structure after strands are exchanged. The RecA protein also promotes branch point migration to resolve this intermediate structure.
Nature also highlights the significant role of the RecA protein by using it as the regulator of the SOS response. (Friedberg, et al., in DNA Repair and Mutagenesis, ASM Press, Washington, D.C. 1995,p. 407) The existence of damaged DNA signals the RecA protein to become activated. This switch promotes the autoproteolysis of the LexA repressor upon binding to a RecA-DNA-ATP complex. Once the LexA repressor is inactivated, a number of protein products are produced, including the RecA protein. Many of these SOS proteins act at the site of DNA damage after the RecA protein has initiated the repair process through recombinational DNA repair. Thus, the RecA protein is an important genomic sentinel for E. coli since it identifies problems and organizes their correctionxe2x80x94all of which ensures the integrity of an organism""s genetic information.
One relatively unexplored region of the RecA protein is the recently defined MAW (Makes ATP Work) motif, located at residues 40-65. It has been proposed that this is part of the conformational switch of the RecA protein that signals the ADP- vs ATP-bound state. It is well established that the RecA protein exhibits different conformations in response to cofactors. (Egelman and Stasiak, Micron 24:309 (1993)).
There are some known mutations of RecA and homologous proteins, within the MAW motif and elsewhere, which relate to either ATP interaction or DNA binding, S. Sommer, F. Boudsocq, R. Devoret, and A. Bailone, Specific RecA Amino Acid Changes Affect RecA-UmuD""C Interaction, Molecular Microbiology 28:281 (1998) discloses a mutation within the MAW motif, substituting leucine for serine at position 44 (44SL) (Mutants are herein denoted by the position number, the letter designation of the removed residue, and the letter designation of the substituted residue; thus, xe2x80x9c44SLxe2x80x9d denotes a serine to leucine switch at position 44). This mutation was isolated randomly through a genetic screen. The in vivo phenotype shows that the mutant behaves like wildtype RecA protein except when it interacts with E. coli protein UmuD""C. Biochemical information regarding the characteristics of 44SL is not available.
P. Howard-Flanders and L. Theriot, Mutants of Escherichia coli K-12 Defective in DNA Repair and in Genetic Recombination, Genetics 53:1137 (1966) discloses a substitution of leucine to phenylalanine at position 51(51LF) within the MAW motif in RecA13. This mutation was shown to be inactive in vivo. Further, S. D. Lauder and S. C. Kowalczykowski, Negative Co-dominant Inhibition of RecA Protein Function: Biochemical Properties of the RecA1, RecA13, and RecA56 Proteins and the Effect of RecA56 Protein on the Activities of the Wild-type RecA Protein Function In Vitro, Journal of Molecular Biology 234:72 (1993) demonstrated that this mutant is inactive in vitro. As discussed below, the substitution occurs at a special location in the MAW structure, and the phenylalanine is insufficiently large to affect the activity of the protein in this position.
In A. J. Clark, The Beginning of a Genetic Analysis of Recombination Proficiency, Journal of Cellular Physiology 70:165 (1967), a RecA56 mutant within the MAW motif is disclosed. This mutant substitutes cysteine for arginine at position 60 (60RC). This mutant was shown in the original disclosure to be inactive in vivo. It has also been shown to be inactive in vitro. See S. D. Lauder and S. C. Kowalczykowski, Negative Co-dominant Inhibition of RecA Protein Function: Biochemical Properties of the RecA1, RecA13, and RecA56 Proteins and the Effect of RecA56 Protein on the Activities of the Wild-type RecA Protein Function In Vitro, Journal of Molecular Biology 234:72 (1993). This substitution is from a volumetrically larger to a smaller residue.
Other mutants in the MAW motif have been disclosed in Ustilago maydis fungi. See B. P. Rubin, D. O. Ferguson, and W. K. Holloman, Structure of REC2, a Recombinational Repair Gene of Ustilago maydis, and Its Function In Homologous Recombination Between Plasmid and Chromosonal Sequences, Molecular and Cellular Biology 14:6287 (1994). This work disclosed five mutants within the MAW motif: rec2-4, substituting alanine for serine at position 42 (42SA); rec2-5, substituting alanine for aspartate at position 48 (48DA); rec2-2, substituting phenylalanine for leucine at position 51 (51LF); rec2-6, substituting alanine for glycine at position 54 (54GA); and rec2-7, substituting alanine for glycine at position 55 (55GA). The 42SA and 55GA mutants involve volumetrically small (alanine) substitutions and behave like the wildtype protein. The 48DA, 51LF, and 54GA mutants are defective in UV damage repair as compared to the wildtype protein. 48DA and 54GA involve volumetrically small substitutions. Although 51LF involves a larger (phenylalanine) substitution, it is located at a geometrically special position in the three-dimensional structure of the MAW motif, and is insufficiently large to enhance the DNA binding properties of the protein.
Other E coil protein mutants are known which bind DNA better than the wildtype protein. See P. E. Lavery and S. C. Kowalczykowski, Biochemical Basis of the Constitutive Repressor Cleavage Activity of RecA730 Protein: A Comparison to RecA441 and RecA803 Proteins, Journal of Biological Chemistry 267:20648 (1992), and M. V. V. S. Madiraju, P. E. Lavery, S. C. Kowalczykowski, and A. J. Clark, Enzymatic Properties of the RecA803 Protein: A Partial Suppressor of recF Mutations, Biochemistry 31:10529 (1992). These publications disclose substitution of methionine for valine at position 37 (37VM) in RecA803, substitution of lysine for glutamate at position 32 (32EK) in RecA441, and substitution of valine for isoleucine at position 298 (298IV) in RecA441. These publicly available RecA803 and RecA441 mutants bind DNA more quickly than wildtype RecA. However, these mutants still require an ATP-like cofactor.
Similarly, the substitution of aspartate for glutamate at position 96 (96ED) in RecA is disclosed in M. J. Campbell and R. W. Davis, On the In Vivo Function of the RecA ATPase, Journal of Molecular Biol. 286:437 (1999). 96ED allows the mutant RecA protein to bind ATP and prevent its hydrolysis, thus keeping the mutant RecA active. However, this mutant is not free of the requirement for the ATP cofactor.
It is desirable to produce a cofactor-independent RecA protein. It is also desirable to produce a RecA protein which binds DNA better than wildtype RecA. The above listed mutants do not meet these goals because they are either inactive, or because they still require a cofactor to activate.
The invention presents RecA homolog protein mutants which have mutations within the MAW motif. The MAW motif is highly conserved among various species, as shown in the sequence analysis of A. I. Roca and M. M. Cox, Progress in Nucleic Acid Research and Molecular Biology 56:129-223 (1997). The MAW motif is defined as amino acid residues 40 to 65 in the Escherichia coli (E. coli) RecA protein (SEQ ID NO: 1) and the homologs of this structure in other proteins. Therefore, as used throughout, the term xe2x80x9cRecA homolog proteinxe2x80x9d refers to an E. coli RecA protein having the MAW sequence at residues 40-65, inclusive, as shown in SEQ ID NO: 1, or a homolog thereof. These homologs include, but are not limited to, homologs in bacteria, viruses, archaea, and eukaryotes (in particular, human). Examples of these RecA homolog proteins are: bacteria (e.g., E. coli) RecA proteins; viruses (e.g., bacteriophage T4) UvsX proteins; Archaea (e.g., Methanococcus jannaschih) RadA proteins; and Eucarya (e.g., Homo sapiens) Rad51, Dmc1, and Lim15 proteins. In each of these RecA homolog proteins, the MAW motif is identified as the structural homolog of the E. coli RecA MAW motif. (See SEQ ID NO: 1; Roca and Cox, supra).
The mutations of this invention modify the MAW region""s properties as an ATP-induced conformational switch and as a DNA binding site of the RecA protein. These mutations involve selectively replacing one or more naturally occurring amino acid residues within the MAW motif with volumetrically larger residues (Class V mutants), or by replacing one or more naturally occurring residues with aromatic residues (Class VI mutants). Class V mutants are mutants which will be active independently of cofactors such as ATP or ATPxcex3S. To achieve this goal, the replacement residue must be sufficiently large, that is, as large or larger than phenylalanine.
Thus, the term xe2x80x9cRecA homolog protein mutantxe2x80x9d as used herein refers to an E. coli RecA protein, or a bacterial, eukaryotic, archaeal, or viral homolog thereof, in which the naturally occurring MAW motif has been modified by one or more such replacements of amino acid residues. xe2x80x9cVolumetrically larger,xe2x80x9d as used herein in reference to a replacement residue, means a residue which is larger than the residue it replaces, and which is as large or larger than phenylalanine.
Because the MAW motif is three-dimensional, the selective positioning of an amino acid residue replacement will affect the physical structure of the MAW motif in a RecA homolog protein mutant. Thus, selective position of such replacements will also affect the behavior of a RecA homolog protein mutant, and this positioning can be controlled to produce particular results. As referred to herein, xe2x80x9cClass V mutantsxe2x80x9d are those mutants which will generally reduce the dependence of the RecA homolog protein mutant on the presence of ATP to initiate DNA binding. Examples of Class V mutants are those with replacements at residues 43, 52, 53, 54, 55, or 59, or combinations thereof. Similarly, xe2x80x9cClass VI mutantsxe2x80x9d are those mutants which will generally bind DNA more tightly than the wildtype RecA homolog proteins from which they are derived. Examples of Class VI mutants are those with aromatic replacements at residues 40, 42, 44, 47, 50, 51, or 56, or combinations thereof. However, the three-dimensional structure of the MAW motif is such that positions 47 and 51 are xe2x80x9cspecialxe2x80x9d sites, requiring a sufficiently volumetrically large aromatic substitution, that is, tryptophan, to meet this goal. Combinations of Class V and Class VI mutations are referred to herein as xe2x80x9cClass VII mutants,xe2x80x9d and will generally exhibit the advantages of Class V and Class VI mutants.
In creating Class V mutants, replacement amino acid residues are selectively volumetrically larger than the wildtype residues which they replace to force structural alterations within the three-dimensional MAW motif, and sufficiently volumetrically large to place the protein in an xe2x80x9copen and activexe2x80x9d state. Therefore, those skilled in the art will recognize that preferable replacement amino acid residues in Class V mutants will be selected from the group of phenylalanine, lysine, tyrosine, arginine, and tryptophan. In creating Class VI mutants, naturally occurring residues are replaced with aromatic residues. Accordingly, those of skill in the art will recognize that preferable replacement amino acid residues in Class VI mutants will be selected from the group of tryptophan, tyrosine, phenylalanine, and histidine. Because of the special nature of positions 47 and 51, it will be recognized that, for these positions, tryptophan is the only aromatic replacement which will be sufficiently large to enhance DNA binding. For example, substitution of tyrptophan for leucine at position 47 (47LW) is active whereas substitution of tyrosine in this position (47LY) is not. This situation is in contrast to that at position 56, where substitution by either tyrosine (56LY) or tryptophan (56LW) is active. (See FIG. 2). In both Class V and VI mutants, tryptophan is the most preferred replacement amino acid residue.
It is an object of this invention to provide RecA homolog protein mutants which are ATP-independent and which can replace RecA in applications which currently require ATP.
It is a further object of this invention to provide RecA homolog protein mutants which provide tighter DNA binding compared to wildtype RecA.